Altered gene expression in embryos and endometrium collected on day 8 of induced aluteal cycles in mares.

Aluteal cycles were induced in the mare to evaluate the effects of progesterone deprivation on the gene expression of embryos and endometrium collected eight days after ovulation. We hypothesized that the transcript expression would be altered during induced aluteal (AL) cycles (low progesterone <1 ng/mL) when compared with control cycles during diestrus (high progesterone; > 4 ng/mL) for 1) the embryonic expression of progesterone-mediated transcripts and those related to normal embryo growth and development and 2) the endometrial expression of progesterone-mediated transcripts and those related to prostaglandin synthesis and normal pregnancy establishment. Seven cyclic mares with a median age of 6.5 years (range 3-16) were utilized in a crossover design. Mares in estrus were artificially inseminated to a fertile stallion and randomly assigned to control or AL groups. Mares received either saline solution (control mares) or PGF2α (AL mares), twice daily on days 0, 1, and 2 and once daily on days 3 and 4. Serial blood samples were collected daily from day 0 (ovulation) until the day of embryo collection and endometrial biopsy on day 8. Mares were monitored until they returned to estrus, and artificially inseminated. Mares were switched to the opposite treatment group only after a successful embryo collection occurred during the previous cycle and only cycles that produced embryos were used for analyses. The study design resulted in paired samples from each mare for analyses. No significant rise in progesterone was observed in the AL group with mean concentrations of plasma progesterone remaining <1.0 ng/mL from ovulation until embryo collection on day 8. This is in sharp contrast to the control (luteal) cycle where a post-ovulatory rise in plasma progesterone was observed. Real-time RT-PCR was utilized to evaluate the expression of ESR1, PGR, CYP19A1, P19, SLC35A1, OCD, APOB, AQP3, NEU2 transcripts in the embryos and PTGS2, P19, ESR1, HK2, sPLA2, PGR, CTGF, IFNE, FGF9, SLC36A2 expression in the endometrium. Four transcripts showed increased expressed in embryos developed during AL cycles ESR1, P19, APOB and PGR (p < 0.05). Four transcripts showed increased expressed in endometrium developed during AL cycles sPLA2, PGR, ESR1, FGF9 (p < 0.05) and four transcripts showed decreased expression P19, CTGF, IFNE, HK2 (p < 0.05). Additionally, staining differences were present in endometrial staining for both ERα and PR receptor during AL cycles compared with control cycles. Embryos and endometrium developed in a progesterone-deprived environment during induced aluteal cycles demonstrated altered transcript expression. These results indicate that adequate progesterone levels may be a key mediator of the appropriate embryo-maternal environment during early preimplantation embryo development.

Morphological evaluation of Day 8 embryos developed during induced aluteal cycles in the mare.

A novel in vivo model utilizing serial administrations of PGF2α to induce aluteal cycles in the mare was used to evaluate the effects of progesterone-deprivation on the morphology of in vivo preimplantation embryos. We hypothesized that equine embryos produced during induced aluteal cycles (AL) would be developmentally affected, characterized by earlier embryo stage at collection, smaller embryo diameter, and lower quality grade, compared with those collected on the same day post-ovulation from control cycles during diestrus (high progesterone; > 4 ng/mL). Seven cyclic mares with a median age of 6.5 years (range 3-16) were utilized in a crossover design. Mares in estrus were artificially inseminated to a fertile stallion and randomly assigned to control or AL groups. Mares received either saline solution (control mares) or PGF2α (AL mares), twice daily on days 0, 1, and 2 and once daily on days 3 and 4. Serial blood samples were collected daily during estrus and until the day of embryo collection 8 days after ovulation. Mares were monitored until they returned to estrus, and artificially inseminated. Mares were switched to the opposite treatment group only after a successful embryo collection occurred during the previous cycle. Only cycles that produced embryos were used for analyses. No significant rise in progesterone was observed in the AL group with mean concentrations of plasma progesterone remaining <1.0 ng/mL from ovulation until embryo collection on Day 8. This is in sharp contrast to the control (luteal) cycle where a post-ovulatory rise in plasma progesterone was observed. The mean daily concentrations of plasma progesterone were significantly higher in control vs. AL group beginning at Day 3 and remained so until Day 8. The mean (±SEM) embryo diameter of AL embryos was 171 ± 5 µm compared to 756 ± 99 µm for control embryos. The majority of the Day 8 AL embryos were classified as morulas (3/9) or early blastocysts (5/9) with only 2 embryos of quality grade 1 compared to the Day 8 control embryos that were mostly expanded blastocysts (6/7) with 5 of 6 being of quality grade 1. This study shows that serial administrations of PGF2α were able to prevent significant rises in plasma progesterone, thus inducing aluteal cycles characterized by a progesterone-deprived environment for developing embryos. Embryos collected from induced aluteal cycles were adversely affected as demonstrated by a lower quality grade, smaller diameter and earlier embryo stage at collection when compared to control embryos.

Biodiesel production using fatty acids from food industry waste using corona discharge plasma technology.

This article aims to describe an alternative and innovative methodology to transform waste, frying oil in a potential energy source, the biodiesel. The biodiesel was produced from fatty acids, using a waste product of the food industry as the raw material. The methodology to be described is the corona discharge plasma technology, which offers advantages such as acceleration of the esterification reaction, easy separation of the biodiesel and the elimination of waste generation. The best conditions were found to be an oil/methanol molar ratio of 6:1, ambient temperature (25 °C) and reaction time of 110 min and 30 mL of sample. The acid value indicates the content of free fatty acids in the biodiesel and the value obtained in this study was 0.43 mg KOH/g. Peaks corresponding to octadecadienoic acid methyl ester, octadecanoic acid methyl ester and octadecenoic acid methyl ester, from the biodiesel composition, were identified using GC-MS. A major advantage of this process is that the methyl ester can be obtained in the absence of chemical catalysts and without the formation of the co-product (glycerin).

Real-time characterization of the uterine blood flow in mares before and after artificial insemination.

The present experiment was divided into two studies to investigate the effect of age and endometrial degeneration on uterine blood flow of mares throughout the immediate post-breeding period. In study 1, uterine blood flow was characterized in mares (n = 7 mares/group) with minimal, moderate or severe endometrial degenerative changes (GI, GII and GIII, respectively). In study 2, the effect of age was investigated using young (≤ 6 years) and old (≥ 15 years) mares (n = 7 mares/group). Uterine vascular perfusion and mesometrial pulsatility index (PI) were evaluated every hour from H0 (moment immediately before AI) to H12. In study 1, a pronounced and transitory increase on uterine vascular perfusion was detected (P < 0.001) between H1 and H3 in the three endometrial groups. In addition, GIII mares had greater (P < 0.05) mesometrial PI than GI mares during the post-breeding period, denoting reduced uterine blood flow. In study 2, a transitory increase on uterine vascular perfusion was also observed in both age groups during the first hour after mating. However, mesometrial PI of young and old mares was similar (P > 0.05) and constant (P > 0.7) through the first 12h after AI. Results demonstrated, for the first time, the immediate changes on uterine vascular perfusion and mesometrial PI in response to semen infusion. Moreover, reduced blood flow of the uterus during the post-breeding period was strongly associated with endometrial degenerative changes in mares, regardless of age.

Effect of age and endometrial degenerative changes on uterine blood flow during early gestation in mares.

The present experiment was divided into three studies to investigate the effect of age and endometrial degeneration on uterine blood flow of mares. In study 1, the influence of semen infusion and conception was evaluated using noninseminated (non-AI), inseminated nonpregnant (AI-NP), and inseminated pregnant (AI-P) mares (n = 7 mares/group). In study 2, the effect of age was investigated using young (≤6 years), adult (from 8 to 12 years), and old (≥15 years) pregnant mares (n = 7 mares/group). In study 3, uterine blood flow was also characterized in pregnant mares (n = 7 mares/group) with minimal, moderate, or severe endometrial degenerative changes (GI, GII, and GIII, respectively). Uterine vascular perfusion and pulsatility index from mesometrium attachment arteries were recorded daily from Day 0 (day of ovulation) to Day 12 and between Days 0 and 20 in nonpregnant and pregnant mares. Analysis according to the position of the embryo (uterine horn with embryo vs. opposite horn) was performed from Day 12 until Day 20. In study 1, increased uterine vascular perfusion and decreased pulsatility index were detected (P < 0.001) between Days 3 and 5 in non-AI and AI-P mares, whereas the uterine vascular perfusion of AI-NP mares did not change (P > 0.05) throughout the experiment. In study 2, the vascular perfusion of the uterine horn with embryo was higher (P < 0.001) than in the opposite uterine horn from Day 12 until Day 20 in both young and adult mares. With exception of Day 15, both uterine horns of old mares showed similar (P > 0.1) vascular perfusion from Day 12. In study 3, vascular perfusion of both uterine horns was lower (P < 0.001) in GIII mares than in GI and GII mares. After Day 15, the scores for uterine vascular perfusion were consistently greater (P < 0.001) in the uterine horn with embryo than in the opposite horn in GI mares. Results showed, for the first time, the reduced blood flow of the gravid uterus during early gestation in older mares and in mares with significant endometrial degeneration.

Antiluteogenic effects of serial prostaglandin F2α administration in cycling mares.

A single dose of PGF2α does not consistently induce luteolysis in the equine CL until at least 5 days after ovulation, leading to the erroneous assumption that the early CL is refractory to the luteolytic effects of PGF2α. We hypothesized that serial administration of PGF2α in early diestrus would induce a return to estrus similar to mares treated with a single injection in mid-diestrus, and fertility of the induced estrus would not differ. The objectives of the study were to evaluate the effects of the 2 approaches as reflected by: (1) concentrations of plasma progesterone; (2) interovulatory and treatment-to-ovulation intervals; (3) the proportion of mares pregnant after artificial insemination. The study consisted of a balanced crossover design in which 10 reproductively normal Quarter Horse Mares were exposed to 2 treatments on 2 consecutive reproductive cycles. At detected ovulation (Day 0), mares were randomly allotted to 1 of 2 treatment groups: I, mid-diestrus treatment, administration of a single 10-mg dose of dinoprost tromethamine (PGF2α) im on Day 10; II, early diestrus treatment, administration of 10-mg PGF2α im twice daily on Days 0, 1, and 2 and once daily on Days 3 and 4. Mares in estrus and with a follicle 35 mm or greater in diameter were artificially inseminated with at least 2 billion motile sperm from a fertile stallion. Pregnancy was defined as detection of a growing embryonic vesicle on 2 consecutive examinations approximately 14 days after ovulation. Serial plasma samples were collected throughout the study period, and concentration of plasma progesterone was determined by RIA. A mixed-model ANOVA for repeated measures was used to analyze hormonal data. Interovulatory and treatment-to-ovulation intervals were compared by a paired t test and fertility by a McNemar chi-square analysis. All mares in group I underwent luteolysis after PGF2α administration denoted by mean (±SD) concentration of plasma progesterone of 0.25 ± 0.21 ng/mL detected 2 days after treatment. In group II, mean concentration of plasma progesterone remained below 1.0 ng/mL during treatment and until the onset of the next estrus. The mean interovulatory interval in group I was 18.5 ± 2.0 days compared with 13.1 ± 3.7 days in group II (P < 0.01). Treatment-to-ovulation intervals were 8.5 ± 2.0 days and 13.1 ± 3.7 days for groups I and II, respectively (P < 0.05). In both groups, 9 of 10 mares were pregnant (P = 1.0). Serial PGF2α administration beginning at ovulation consistently prevented luteal function in 10 of 10 mares in the present study without adversely affecting pregnancy rate of post-treatment cycles.

Paediatric community-acquired septic shock: results from the REPEM network study.

UNLABELLED: INTRODUCTION AND PURPOSE OF THE STUDY: With this study we aimed to describe a "true world" picture of severe paediatric 'community-acquired' septic shock and establish the feasibility of a future prospective trial on early goal-directed therapy in children. During a 6-month to 1-year retrospective screening period in 16 emergency departments (ED) in 12 different countries, all children with severe sepsis and signs of decreased perfusion were included. RESULTS: A 270,461 paediatric ED consultations were screened, and 176 cases were identified. Significant comorbidity was present in 35.8 % of these cases. Intensive care admission was deemed necessary in 65.7 %, mechanical ventilation in 25.9 % and vasoactive medications in 42.9 %. The median amount of fluid given in the first 6 h was 30 ml/kg. The overall mortality in this sample was 4.5 %. Only 1.2 % of the survivors showed a substantial decrease in Paediatric Overall Performance Category (POPC). 'Severe' outcome (death or a decrease ≥2 in POPC) was significantly related (p < 0.01) to: any desaturation below 90 %, the amount of fluid given in the first 6 h, the need for and length of mechanical ventilation or vasoactive support, the use of dobutamine and a higher lactate or lower base excess but not to any variables of predisposition, infection or host response (as in the PIRO (Predisposition, Infection, Response, Organ dysfunction) concept). CONCLUSION: The outcome in our sample was very good. Many children received treatment early in their disease course, so avoiding subsequent intensive care. While certain variables predispose children to become septic and shocked, in our sample, only measures of organ dysfunction and concomitant treatment proved to be significantly related with outcome. We argue why future studies should rather be large multinational prospective observational trials and not necessarily randomised controlled trials.

Effect of intrauterine dextrose or antibiotic therapy on reproductive performance of lactating dairy cows diagnosed with clinical endometritis.

The objectives of this study were to assess the responses to treatments (clinical cure and cow survival 14 d posttherapy) of cows with clinical endometritis (CE) that received intrauterine infusion of a hypertonic solution of 50% dextrose (DEX) or subcutaneous ceftiofur crystalline free acid (CCFA) and subsequent pregnancy per artificial insemination (P/AI) in cows with CE compared with cows without CE. Cows (n=760) from 2 dairy herds were screened for CE using vaginoscopy and measurement of cervix diameters [exam 1; 26±3 d in milk (DIM)]. Cows with vaginal discharge scores of 2 or 3 (scale 0-3) were stratified by parity and randomly allocated into 1 of 3 treatment groups: (1) intrauterine infusion (∼200 mL) of 50% DEX solution (n=79); (2) 6.6 mg/kg single-dose of subcutaneous administration of CCFA (n=75); or (3) untreated control animals (CON, n=83). Fourteen days posttherapy (at 40±3 DIM), cows with CE were re-examined (exam 2; 40±3 DIM) to assess the response to treatments. All cows were presynchronized with 2 injections of PGF(2α) given 14 d apart (starting at 26±3 DIM) followed by Ovsynch (OV; GnRH-7 d-PGF-56 h-GnRH 16 h-timed-AI) 12 to 14 d later. Cows displaying signs of standing estrus any time during the protocol were inseminated, whereas the remaining cows were subjected to timed AI 16 h after the second GnRH of OV. Pregnancy diagnosis was performed via transrectal ultrasonography at 39±3d post-AI followed by pregnancy reconfirmation 30 d after the first pregnancy diagnosis. Uterine swabs revealed that Arcanobacterium pyogenes and Escherichia coli were the most predominant bacteria isolated at the time of treatments. Mortality within 14 d posttherapy was not different among treatment groups. Cows with CE had greater cervical diameter at exam 1 and decreased P/AI compared with cows without CE. Treatment with CCFA or DEX increased the proportion of cows with clear vaginal discharge (score 0; clinical cure) 14 d posttherapy compared with CON cows. Pregnancy per AI from DEX (29.8±4%) cows tended to differ from that of CON (21.1±4%) or CCFA cows (19.7±4%), but it resulted in similar P/AI as those cows without CE (39.1±2%). The use of intrauterine DEX alone or as an adjunct of antibiotic therapy for the treatment of CE needs further investigation.

Rescue administration of a helper-dependent adenovirus vector with long-term efficacy in dogs with glycogen storage disease type Ia.

Glycogen storage disease type Ia (GSD-Ia) stems from glucose-6-phosphatase (G6Pase) deficiency and causes hypoglycemia, hepatomegaly, hypercholesterolemia and lactic acidemia. Three dogs with GSD-Ia were initially treated with a helper-dependent adenovirus encoding a human G6Pase transgene (HDAd-cG6Pase serotype 5) on postnatal day 3. Unlike untreated dogs with GSD-Ia, all three dogs initially maintained normal blood glucose levels. After 6-22 months, vector-treated dogs developed hypoglycemia, anorexia and lethargy, suggesting that the HDAd-cG6Pase serotype 5 vector had lost efficacy. Liver biopsies collected at this time revealed significantly elevated hepatic G6Pase activity and reduced glycogen content, when compared with affected dogs treated only by frequent feeding. Subsequently, the HDAd-cG6Pase serotype 2 vector was administered to two dogs, and hypoglycemia was reversed; however, renal dysfunction and recurrent hypoglycemia complicated their management. Administration of a serotype 2 HDAd vector prolonged survival in one GSD-Ia dog to 12 months of age and 36 months of age in the other, but the persistence of long-term complications limited HDAd vectors in the canine model for GSD-Ia.

Effect of season on fresh and cryopreserved stallion semen.

The objective of this study was to determine the effect of season on sperm quality variables, expression of the fertility-related protein SP22 and selected mRNA transcripts in fresh and cryopreserved stallion sperm. Four stallions were collected in each of the four seasons: summer, fall, winter and spring. Ejaculates were divided and then evaluated for motility, morphology, SP22 staining and expression of selected mRNAs as either fresh semen samples or cryopreserved samples. A significant interaction between season and cryopreservation status was found for total and progressive sperm motility. RNA yield from sperm was not affected by any variable examined. There was no effect of season or cryopreservation on the relative amounts of mRNA for PGK2, TPX1, TIMP3 or ACTB. There was a tendency (P=0.1) for an effect of stallion on the relative amount of ACTB mRNA. The proportion of sperm immunostained for SP22 over the equatorial segment was affected (P<0.05) by stallion. In addition, there was an interaction (P<0.05) between season and cryopreservation status on the percentage of sperm staining for SP22 on the equatorial segment. The correlation among total motility, progressive motility and SP22 immunostaining was much greater (P<0.05) during the breeding season (March and June) than during the non-breeding season (September and December). Based on data analyzed, semen collected in the Northern Hemisphere between March and June may be best suited for cryopreservation.

Pharmacokinetics of carbetocin, a long-acting oxytocin analogue, following intravenous administration in horses.

REASON FOR PERFORMING STUDY: Current therapy protocols to treat persistent post mating endometritis and retained fetal membranes in mares typically include the administration of ecbolic drugs. Evaluation of the pharmacokinetics and tolerability of carbetocin, a long-acting oxytocin analogue, after i.v. administration is required. OBJECTIVES: To determine the pharmacokinetic parameters (principally half-life) of carbetocin in horses. METHODS: Five mature mares and one gelding received 0.175 mg carbetocin i.v. All animals were monitored periodically throughout the study for elevation in rectal temperature, heart rate, respiratory rate and signs of pain or discomfort. Plasma samples were collected for determination of carbetocin concentrations by radioimmunoassay. RESULTS: Administration of carbetocin was well tolerated by all horses and its half-life was 17.2 min. CONCLUSIONS: The half-life of carbetocin is greater than that previously reported for oxytocin (6.8 min). POTENTIAL RELEVANCE: Carbetocin is an attractive alternative to oxytocin therapy in broodmare management.

Simplified hypoosmotic swelling testing (HOST) of fresh and frozen-thawed canine spermatozoa.

The clinical use of the hypoosmotic swelling test (HOST) to identify spermatozoa with a functional intact membrane has been reported for humans and domestic species, including the dog. Currently, it is recommended that canine spermatozoa be incubated with the hypoosmotic solution for periods that range from 30 to 60 min. In an attempt to simplify the test, it was hypothesized that the degree of the hypoosmotic response at 1 min of incubation would not be different from the response documented at 60 min after incubation in the hypoosmotic solution at 37 degrees C. The hypoosmotic response of spermatozoa from 50 fresh and 16 frozen-thawed semen samples obtained from 22 adult dogs was recorded at 1 and 60 min of incubation. There were no significant differences between the hypoosmotic response recorded at 1 and 60 min for all evaluated semen samples (P>0.10). The hypoosmotic response recorded for canine spermatozoa from fresh semen samples were greater than that recorded for spermatozoa from frozen-thawed semen, both at 1 min (86.2% compared with 65.2%; P<0.001) and 60 min (85.6% compared with 61.8%; P<0.001). Based on the results of this study, it is recommended to decrease the incubation time of the HOST for canine spermatozoa to as short a period as 1 min. This incubation time should encourage the application of this relatively simple and inexpensive test of canine sperm membrane function in a clinical setting.

Successful induction of immune tolerance with FIX recombinant in a patient with haemophilia B with inhibitor.

We describe successful induction of immune tolerance in a 12-month-old patient with severe haemophilia B and low-titre and low response antifactor IX inhibitor at 5 Bethesda Unit (BU). Immune tolerance induction (ITI) was started with recombinant factor (rFIX) at 40-50 U kg(-1) day(-1) (1000 U three times per week) and 2 years after the beginning of therapy the inhibitor had been reduced to undetectable values. As complications, which appeared during the ITI, the patient presented minor haemorrhagic complications, which remitted following the administration of rFVIIa, and recurring infections of the catheter, requiring the withdrawal and subsequent placement of a new catheter.

Concentrations of nitric oxide in equine preovulatory follicles before and after administration of human chorionic gonadotropin.

In the present study, follicular fluids of estrous mares treated with saline solution (Control) or nitric oxide synthase (NOS) inhibitors were analyzed for nitric oxide (NO), estradiol-17beta (E2) and progesterone (P4) concentrations before and 36h after administration of human chorionic gonadotropin (hCG). Follicular fluids obtained before (0h) hCG administration from control mares had lower concentrations of NO than those obtained 36h after administration of hCG (58.3+/-17.8 micromol versus 340.4+/-57.7 micromol; P<0.05). A similar pattern was also noted for intrafollicular P4 in control mares, which had lower concentrations of intrafollicular P4 before hCG than 36h post-hCG administration (P<0.05). As expected, E2 concentrations of control follicles sampled before hCG administration were higher than those sampled 36h post-hCG administration (P<0.05). However, the E2 concentrations in follicles of mares treated with the NOS inhibitors N(omega)-nitro-L-arginine methyl ester (L-NAME) or aminoguanidine (AG) did not decrease after hCG administration, unlike those in control mares (P>0.10). In addition, mares treated with NOS inhibitors had lower intrafollicular concentrations of NO and P4 than control mares, both before and after hCG administration (P<0.05). Increased intrafollicular concentrations of NO in control, hCG-stimulated mares provide evidence for the presence of an NO-generating system in the equine preovulatory follicle that is likely upregulated following administration of hCG.

Day of cycle affects changes in equine intrauterine pressure in response to teasing.

Oxytocin is released in response to teasing during both estrus and diestrus in mares, and at least during estrus, teasing results in an increase in electromyographic activity in the uterus. Exogenous oxytocin causes an increase in intrauterine pressure and prior studies have shown that this response is correlated to the day of the estrous cycle. To determine if teasing causes an increase in intrauterine pressure and if this response varies by day of the cycle, intrauterine pressure was measured while mares were teased with a stallion 2 days before ovulation, on the day ovulation was detected and 2 days after ovulation. A significant increase in intrauterine pressure was observed in response to teasing both 2 days before ovulation and on the day of ovulation, when plasma concentrations of progesterone were low. No significant increase in intrauterine pressure was observed in response to teasing 2 days after ovulation when progesterone concentrations were elevated. Management practices that include teasing or stallion exposure may be beneficial in stimulating uterine clearance mechanisms in mares during the preovulatory period.

Effect of nitric oxide synthase inhabitors on ovulation in hCG-stimulated mares.

Recent studies suggest that nitric oxide (NO) may have a role in regulating ovarian physiology. To investigate the role of NO during ovulation in mares, inhibitors of the nitric oxide synthase (NOS) were administered to estrous mares. Forty cycling mares (20 horses and 20 pony mares) were allotted to one of the three treatment groups. Once a follicle was at least 27 mm in diameter, but smaller than 35 mm, mares were given one of the following treatments: saline solution 0.9% (n = 20, w/v, i.v., every 12 h), Nomega-nitro-L-arginine methyl ester hydrochloride (L-NAME; n = 10, 148 micromol/kg, i.v., every 12 h), or aminoguanidine hemisulfate (AG; n = 10, 406 micromol/kg, i.v., every 12 h). When a follicle >30 mm was present on one of the ovaries, ovulation was induced with hCG (2,500 IU, i.v.). The median time of ovulation (+/-6 h) after hCG administration for the treatment groups was 42, 84 and 54 h for mares treated with saline solution, L-NAME and AG, respectively. There was no significant difference between the groups treated with AG or L-NAME (P = 0.06); however, these groups were different from the control group (P < 0.05). The delayed ovulation caused by the administration of NOS inhibitors suggests a role for NO in follicular growth and ovulation in horses.

The prothrombin 20210A allele and the factor V Leiden are associated with venous thrombosis but not with early coronary artery disease.

This study was performed in order to establish the role of the prothrombin 20210 G/A and factor V Leiden (R506Q) polymorphisms in the susceptibility to develop venous thromboembolism and early coronary artery disease (CAD). These polymorphisms were determined in 82 consecutive patients with venous thromboembolism, 175 male patients with early CAD, and 200 healthy controls from the same Caucasian population (Asturias, Northern Spain). DNA was amplified using polymerase chain reactions and digested with the appropriate restriction enzymes in order to define the prothrombin and factor V genotypes. The prevalence of the heterozygous for the prothrombin A allele was 3.5% in the general population and 15.8% in patients with venous thrombosis (P = 0.0007); the frequency was 4% in patients with early CAD. No sex-related differences in the prevalence of the A allele were observed, and the average age at the first venous thromboembolic event was similar between GG and AG patients. The frequency of carriers of the factor V Leiden polymorphism was 9.75% among patients with venous thromboembolism, compared with 3.5% among controls, and 3.4% in the patients with CAD. Our data showed an association between venous thromboembolism and the AG genotype at the prothrombin 20210 G/A polymorphism. This polymorphism was not related to an increased risk for early CAD in our population of male patients.